Methods for modifying human antibodies by glycan engineering

ABSTRACT

Modified Fc regions of antibodies and antibody fragments, both human and humanized, and having enhanced stability and efficacy, are provided. Fc regions with core fucose residues removed, and attached to oligosaccharides comprising terminal sialyl residues, are provided. Antibodies comprising homogeneous glycosylation of Fc regions with specific oligosaccharides are provided. Fc regions conjugated with homogeneous glycoforms of monosaccharides and trisaccharides, are provided. Methods of preparing human antibodies with modified Fc using glycan engineering, are provided.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This patent application claims the benefit or priority of, and is a Continuation of, U.S. application Ser. No. 12/959,351, filed Dec. 2, 2010, which claims priority of U.S. Provisional Patent Application Ser. No. 61/265,757, filed Dec. 2, 2009, entitled “Methods for Modifying Human Antibodies by Glycan Engineering” the contents of each of which is hereby incorporated by reference in its entirety.

TECHNICAL FIELD OF THE INVENTION

This invention relates to the field of antibodies. In particular, the application relates to methods for modifying glycosylation states of human monoclonal antibodies (mAbs) by glycan engineering. More particularly, the application relates to uniformly glycosylated glycoforms of Fc regions of human mAbs and methods for preparing the antibodies by glycan engineering.

BACKGROUND OF THE INVENTION

Antibodies, also known as immunoglobulins (Ig), are glycoproteins that play a central role in immune responses. IgG antibodies (Abs) and fragments of IgG Abs have become major biotherapeutics for treating human diseases.

There are five functional classes of immunoglobulins, i.e., immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin G (IgG), immunoglobulin A (IgA) and immunoglobulin E (IgE). Among them, IgG is the most abundant immunoglobulin in serum.

The variable region (Fab) of the antibody molecule is involved in direct binding of the target antigen. All naturally-occurring antibodies each include a constant domain known as the Fc (Fragment, crystallizable) region, which is composed of constant domains depending on the class of the antibody. By binding to specific proteins the Fc region ensures that each antibody generates an appropriate immune response for a given antigen. The Fc region also binds to various cell receptors, such as Fc receptors, and other immune molecules, such as complement proteins. By doing this, it mediates different physiological effects including opsonization, cell lysis, and degranulation of mast cells, basophils and eosinophils. (Woof J, Burton D (2004) Nat Rev Immunol 4 (2): 89-99; Heyman B (1996) Immunol. Lett. 54 (2-3): 195-199). Glycosylation of the antibody Fc fragment is essential for Fc receptor-mediated activity. (Peipp M. et al., Blood (2008) 112(6):2390-2399).

The constant (Fc) domain of naturally-occurring antibodies is usually N-glycosylated. The linkage of carbohydrates to proteins occurs through N-linked glycosylation—the attachment of a sugar to the amide nitrogen atom on the side chain of asparagines. The linkage to amino acids is generally embedded in a conserved sequence of amino acids. Glycosylation in the Fc region significantly affects Fc effector functions such as activation of complement (Roos et al., J Immunol. (12):7052-7059.2001) or binding to receptors via their constant (invariable sequence) domains (Mimura et al., J Biol. Chem. 2001 Dec. 7; 276(49):45539-45547; Shields et al., J Biol. Chem. 276(9):6591-6604 (2001)).

It has been found that the sequences of the oligosaccharides attached to Fc regions are essential to the stability and function of the antibodies. For example, the addition of sialic acid (SA), sialylation, at Asn 297 on the Fc domain and terminal galactosylation modify the anti-inflammatory properties of immunoglobulins and play a role in rheumatoid arthritis (RA). (Kaneko, Yet al., Science. (2006) 313(5787):670-673).

Human antibodies prepared by conventional methods, such as genetic engineering, comprise mixtures of different glycoforms, i.e., containing different oligosaccharides attached to their Fc regions.

The clinical successes of the antibodies rituximab and alemtuzumab for hematological malignancies, and trastuzumab for solid breast tumors, have demonstrated that human or humanized monoclonal antibodies can be useful drugs for the treatment of cancer. All of these antibodies have been approved and are currently in the market. Yet, a need remains for improving this kind of drugs. For instance, rituximab induces a response only in approximately 60% of the patients with relapsed/refractory low-grade non-Hodgkin's lymphoma.

The preferred type of monoclonal antibody for target-cell killing applications is an IgG1-type mAb, where at least the constant (or the Fc) region is of human origin (i.e., chimeric, humanized, or fully-human IgG1s). IgG1 mAbs can exert their therapeutic effects via three classes of mechanisms. Direct binding of the antibody via its variable region to the target molecule on the surface of cancer cells can lead to cell death or inhibition of tumor growth, for example by triggering apoptosis upon cross-linking of target molecules by the antibody. The Fc region operates in the other two-types of mechanisms: complement-mediated cytotoxicity, and Fcγ receptor (FcγR)-dependent effector functions. Some Fc effector functions of IgG1s are mediated by the interactions of the Fc region of target-cell bound antibody molecules and Fcγ receptors on the surface of immune cells. The Fc domains of immunoglobulins have been shown to have effector functions which are primarily complement fixation and Fc receptor (FcR) binding. appropriate glycosylation at the conserved glycosylation site (N297) of the Fc domain is essential for the efficient interactions between mAbs and Fc receptors (FcR) and for the FcR-mediated effector functions, including antibody-dependent cell mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Fc receptors bind to the constant domains of immunoglobulins and a number of receptors have been defined that are thought to mediate accessory functions including opsonization and ADCC (Daeron, M. Annual Review Immunology, (1997) 15; 203-234, Ravetch and Clynes, Annual Review of Immunology. 1998. 16:421-432).

The conserved glycosylation site on the Fc region of antibodies is a target for modulation of antibody effector functions. The crystal structure of a biosynthetic intermediate of human IgG1, bearing immature oligomannose-type glycans and reported to display increased antibody-dependent cellular cytotoxicity, has shown that glycan engineering can bias the Fc to an open conformation primed for receptor binding. (Crispin M., et al., J. Mol. Biol. 387(5):1061-1066 (2009).)

More specifically, certain glycoforms of a human antibody exhibit improved therapeutic effects while others possess undesired properties. For example, de-fucosylated but glycosylated Herceptin® is at least 50-fold more active in the efficacy of Fcγ receptor IIIa mediated ADCC than those with alpha-1,6-linked fucose residues (Shields, R L, et al. J. Biol. Chem., Vol. 277(30):26733-26740, (2002)). Similar results were reported for Rituximab® and other mAbs (Shinkawa T, et al., J. Biol. Chem. (2003) 278:3466-3473).

Strome et al., (WO 2007/146847) disclose a method of preparing antibodies with homogeneous glycosylation states by first deglycosylating an antibody or antibody fragment and then attaching a chemically synthesized sugar to the protein. This method lacks the ability to control the glycosylation process and does not enable control over the conjugation of specific carbohydrates at specific sites during the formation of a desired glycoform. Thus, there is a need for methods of preparing human antibodies comprising desired glycoform(s) wherein the glycosylation is performed in a stepwise manner so as to improve their therapeutic efficacy.

SUMMARY OF THE INVENTION

The invention relates to the Fc region of an antibody molecule, wherein the Fc region is specifically glycosylated with oligosaccharides that increase the efficacy and stability of the Fc region, and the antibody or antibody fragment comprising the Fc region. In some embodiments the specifically glycosylated Fc fragment comprises a monoclonal antibody, preferably a human or humanized monoclonal antibody.

In one aspect, the present invention features an antibody in which both of the Ig domains are attached in the Fc region to oligosacchardes that are free of core fucoses and/or contain terminal sialic acids linked to galactoses. In some embodiments, the oligosaccharides are homogeneous. In some embodiments, the antibody is human or humanized. In some embodiments, the Fc region comprises an antibody or an antibody fragment.

In one embodiment, the terminal sugar units of the invention are sialic acids linked to galactose. N-Acetyl-Neuraminic Acid (Neu5Ac or NANA) is the predominant sialic acid found in mammalian cells. When the human antibody is an IgG, each of its Ig domains can comprise an Fc region containing an N-linked biantenary oligosaccharide with two terminal sialic acids each linked to a galactose, e.g., an oligosaccharide having the structure:

In another aspect, the present invention relates to Fc regions within an antibody or antibody fragment in which both of the Ig domains comprise Fc regions that are attached to either a monosaccharide moiety (e.g., N-Acetylglucosamine, GlcNAc) or a trisaccharide moiety (e.g., Mannose-N-Acetylglucosamine-N-Acetylglucosamine, Man-GlcNAc-GlcNAc).

The invention further relates to methods for preparing the antibodies and antibody fragments disclosed herein.

The invention relates to a method for modifying the Fc region glycosylation pattern of a human or humanized antibody, the method comprising: (a) providing a glycosylated Fc region of a human or humanized antibody; (b) contacting the glycosylated Fc region with alpha-fucosidase under conditions suitable for removing core fucose residues; (c) contacting the de-fucosylated Fc region with β-1,4-galactosyltransferase under conditions suitable for attaching a galactose to the oligosaccharaide; and then (d) contacting the Fc region with alpha-2,6-sialytransferase under conditions suitable for attaching a terminal sialyl residue, thereby producing a modified Fc region of an antibody. When both β-1,4-galactosyltransferase and alpha-2,6-sialyltransferase are used, the antibody to be modified is first contacted with the β-1,4-galactosyltransferase and then the alpha-2,6-sialyltransferase. In some aspects, one or more of alpha-fucosidase, β-1,4-galactosyltransferase and alpha-2,6-sialytransferase are immobilized on a solid support.

In another aspect, the method comprises: (i) providing a Fc region of an antibody or antibody fragment, wherein the Fc region is glycosylated with an oligosaccharide; (ii) contacting the Fc region with an endo-glycosylase (such as endo-H, endo-F3, etc.) and an exo-glycosylase (such as sialidase, galactosidase, alpha-fucosidase, or a mixture thereof) under conditions wherein the oligosaccharide is digested to a single sugar unit (such as GlcNAc); (iii) elongating the single sugar unit to an oligosaccharide by glycosylation mediated by one or more glycosyltransferases such as endo-N-acetylglucosaminidase, endo-M or endoA; and (iv) contacting the oligosaccharide with alpha-2,6-sialytransferase to add a terminal sialic acid, thereby yielding the antibody having homogeneous oligosaccharides.

In some aspects, the oligosaccharide obtained from the elongating step has a sequence of sugars that is identical to at least a portion of an oligosaccharide comprising a glycosylated Fc region of a naturally-occurring antibody, for example an Ig molecule produced in vivo (either in cell culture or in a host animal). When the antibody is an IgG, the oligosaccharide can have the structure:

The invention further relates to a method for modifying the Fc region glycosylation pattern of a human or humanized antibody, the method comprising: (i) providing a Fc region of an antibody or antibody fragment, wherein the Fc region is glycosylated with an oligosaccharide; (ii) contacting the Fc region with an endo-glycosylase (e.g., endo-H, endo-F3, etc.) and an exo-glycosylase (e.g., sialidase, galactosidase, alpha-fucosidase, or a mixture thereof) under conditions wherein the oligosaccharide is digested to a single sugar unit (e.g., GlcNAc); (iii) elongating the single sugar unit to a first oligosaccharide via glycosylation mediated by glycosyltransferases such as endo-N-acetylglucosaminidase, endo-M or endo-A; (iv) contacting the first oligosaccharide with a β-1,4-galactosyltransferase to produce a second oligosaccharide having a terminal galactose, and (v) contacting the second oligosaccharide with alpha-2,6-sialytransferase to add a terminal sialic acid, thereby yielding an antibody having homogeneous oligosaccharides.

In another embodiment, the method comprises: (i) providing a Fc region of an antibody or antibody fragment, wherein the Fc region is glycosylated with an oligosaccharide; (ii) contacting the Fc region with an exo-glycosylase (e.g., alpha-mannosidase, alpha-fucosidase, sialidase, galactosidase, or a mixture thereof) under conditions wherein the oligosaccharides are trimmed to a trisaccharide (e.g., ManGlcNAcGlcNAc); (iii) elongating the trisaccharide to an oligosaccharide via glycosylation; and (iv) treating the oligosaccharide with alpha-2,6-sialytransferase to add terminal sialic acids, thereby yielding Fc regions of the antibody population in a single glycoform.

In some embodiments, the Fc region comprises a human IgG sequence and the oligosaccharide has the structure:

The oligosaccharide is then subjected to alpha-2,6-sialytransferase treatment to add terminal sialic acid residues.

The invention relates to a method for modifying the Fc region glycosylation pattern of a human or humanized antibody, the method comprising: (a) providing a glycosylated Fc region of a human antibody; (b) contacting the glycosylated Fc region with an endo-glycosylase and an exo-glycosylase under conditions wherein the native oligosaccharide is digested to a single sugar unit; (c) contacting the single sugar glycosylated Fc region with a glycosyl transferase under conditions suitable for elongating the single sugar unit to a first oligosaccharide by glycosylation; (d) contacting the first oligosaccharide with β-1,4-galactosyltransferase to produce a second oligosaccharide having a terminal galactoses; and (e) contacting the oligosaccharide with alpha-2,6-sialytransferase under conditions suitable for adding a terminal sialylic acid, thereby producing a modified glycosylated Fc region.

In some embodiments, the Fc region comprises a human IgG sequence and the first oligosaccharide has the structure of:

The invention further relates to a method for modifying the Fc region glycosylation pattern of a human or humanized antibody, the method comprising: (a) providing a glycosylated Fc region of a human antibody; (b) contacting the glycosylated Fc region with an endo-glycosylase and an exo-glycosylase under conditions wherein the oligosaccharide is digested to a single sugar unit; (c) contacting the single sugar glycosylated Fc region with a glycosyl transferase under conditions suitable for elongating the single sugar unit to an oligosaccharide by glycosylation; and (d) contacting the oligosaccharide with alpha-2,6-sialytransferase under conditions suitable for adding a terminal sialylic acid, thereby producing a modified glycosylated Fc region.

In some embodiments, the Fc comprises a human IgG and the trisaccharide is ManGlcNAcGlcNAc, wherein the terminal GlcNAc unit of the trisaccharide is attached to the Fc region of the antibody.

In some embodiments, the Fc region comprises a human IgG sequence and the oligosaccharide has the structure:

In some embodiments, the elongating step is performed by contacting the trisaccharide sequentially with mannosyltransferase, GlcNAc transferase, and galacosyltransferase.

In some aspects, the endo-glycosidase is selected from the group consisting of: endo-beta-N-acetylglucosaminidases (NAG), endo-H, endo-A and endo-M.

In some aspects, the exo-glycosidase is selected from the group consisting of: s alpha-mannosidase, alpha-fucosidase, sialidase, galactosidase, and a mixture thereof.

In some aspects, the oligosaccharide produced by the elongating step has a sugar sequence identical to an oligosaccharide correlated with efficient FcR-mediated effector functions. In some embodiments thereof, the FcR-mediated effector function is selected from antibody-dependent cell mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

The invention further relates to an antibody or antibody fragment comprising a modified Fc region glycosylation pattern, wherein the Fc region is prepared by any method disclosed herein. In some aspects, the human or humanized antibody is selected from the group consisting of: Reopro®, Rituxan®, Zenepax®, Simulect®, Synagis®, Remicade®, Herceptin®, Mylotarg®, Campath®, Zevalin®, Humira®, Xolair®, Bexxar®, Raptiva®, Erbitux®, Avastin®, tysabri®, a monoclonal antibody, a human or humanized antibody undergoing clinical trials for therapeutic use, and a human or humanized antibody produced by a conventional method.

In some embodiments the invention comprises a plurality of antibody or antibody fragments according to any of the methods discosed herein, wherein the Fc regions are uniformly glycosylated with the same oligosaccharide sequence.

In another aspect, the invention relates to a human or humanized antibody comprising Fc regions glycosylated at the Fc region with a single glycoform, wherein the antibody is produced by: (a) providing a human or humanized antibody comprising Fc regions glycosylated with heterogeneous glycoforms; and (b) treating the Fc region sequence with a mannosidase and a sialidase under conditions wherein only a trisaccharide is retained at the glycosylation site. In some embodiments, the trisaccharide is ManGlcNAcGlcNAc, and the trisaccharide is conjugated to the Fc region via the terminal GlcNAc residue.

In some aspects, the invention relates to an isolated human antibody, wherein its Fc region is attached to specifically conjugated trisaccharides and are free of other oligosaccharides. In one embodiment, the human antibody is an IgG and at least one of the trisaccharides is ManGlcNAcGlcNAc, in which the terminal GlcNAc unit is attached to the Fc region of the IgG.

In some aspects, the invention relates to a polypeptide comprising the Fc region sequence of an antibody molecule, wherein the polypeptide comprises an N-glycosylation site of the Fc molecule, and further wherein the glycosylation site is conjugated to a monosaccharide, such as GlcNAc. In some aspects, the Fc glycosylation site comprises an asparagine (Asn, N) amino acid residue. In some aspects, the Fc region comprises an IgG antibody and the N-glycosylation site is Asn-297 residue. In some embodiments, the polypeptide comprises an antibody, or a human or humanized antibody. In some embodiments, the human or humanized antibody comprises Fc regions glycosylated with a single glycoform comprising the monosaccharide, GlcNAc.

In some aspects, the polypeptide comprises an N-glycosylation site of the Fc molecule, and further wherein the glycosylation site is conjugated to a trisaccharide. In some embodiments, the trisaccharide is ManGlcNAcGlcNAc, and the trisaccharide is conjugated to the Fc region via the terminal GlcNAc residue.

In some aspects, the invention relates to a human or humanized antibody comprising Fc regions glycosylated at the Fc region with a single glycoform, wherein the antibody is produced by: (a) providing a human or humanized antibody comprising Fc regions glycosylated with heterogeneous glycoforms; and (b) treating the Fc region sequence with a galactosidase and a sialidase under conditions wherein only a monosaccharide is retained at the glycosylation site. In some embodiments, the monosaccharide is GlcNAc. In some embodiments, the human or humanized antibody comprises Fc regions glycosylated with a single glycoform comprising the trisaccharide, ManGlcNAcGlcNAc.

The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawing and detailed description of several embodiments, and also from the appended claims.

DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, the inventions of which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 shows a schematic illustration depicting a pathway (Path A) for modifying a human antibodies by glycan engineering, using alpha-fucosidase, alpha-2,6-sialyltransferase, and optionally β-1,4-galactosyltransferase.

FIG. 2 shows a schematic illustration depicting a chemoenzymatic pathway (Path B) for preparing human antibodies in a single glycoform by glycan engineering, using various exo-glycosidases, endo-glycosidases, and glycosyltransferases.

FIG. 3 shows a schematic illustration depicting an enzymatic pathway (Path C) for preparing human antibodies in a single glycoform by glycan engineering, using various exo-glycosidases and glycosyltransferases.

FIG. 4 shows a schematic illustration depicting the regeneration of the cofactor CMP-Neu5Ac and UDP-galactose.

FIG. 5A shows a schematic illustration of digestion of heterogeneous glycoforms of Humira® obtained from Chinese hamster ovary (CHO) cells with Endo F2 and Endo F3. The results are shown in Coomassie stained polyacrylamide gels in FIG. 5B.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are modified human antibodies prepared via glycan engineering. The invention relates to the Fc region of an antibody molecule, wherein the Fc region is specifically glycosylated with oligosaccharides that increase the efficacy and stability of the Fc region, and the antibody or antibody fragment comprising the Fc region. In some embodiments the specifically glycosylated Fc fragment comprises a monoclonal antibody, preferably a human or humanized monoclonal antibody. Methods for generating such Fc glycosylated antibodies or antibody fragments by glycan engineering are disclosed herein

In the following description, reference is made to the accompanying drawings that form a part hereof, and in which is shown by way of illustration specific embodiments which may be practiced. These embodiments are described in detail to enable those skilled in the art to practice the invention, and it is to be understood that other embodiments may be utilized and that structural, logical and electrical changes may be made without departing from the scope of the present invention. The following description of example embodiments is, therefore, not to be taken in a limited sense, and the scope of the present invention is defined by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications and patents specifically mentioned herein are incorporated by reference for all purposes including describing and disclosing the chemicals, cell lines, vectors, animals, instruments, statistical analysis and methodologies which are reported in the publications which might be used in connection with the invention. All references cited in this specification are to be taken as indicative of the level of skill in the art. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

Before the present materials and methods are described, it is understood that this invention is not limited to the particular methodology, protocols, materials, and reagents described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

Definitions

It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. As well, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein. It is also to be noted that the terms “comprising”, “including”, and “having” can be used interchangeably.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Antibodies: A Laboratory Manual, by Harlow and Lane s (Cold Spring Harbor Laboratory Press, 1988); and Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986).

As used herein, the term “glycan” refers to a polysaccharide, or oligosaccharide. Glycan is also used herein to refer to the carbohydrate portion of a glycoconjugate, such as a glycoprotein, glycolipid, glycopeptide, glycoproteome, peptidoglycan, lipopolysaccharide or a proteoglycan. Glycans usually consist solely of O-glycosidic linkages between monosaccharides. For example, cellulose is a glycan (or more specifically a glucan) composed of β-1,4-linked D-glucose, and chitin is a glycan composed of β-1,4-linked N-acetyl-D-glucosamine. Glycans can be homo or heteropolymers of monosaccharide residues, and can be linear or branched. Glycans can be found attached to proteins as in glycoproteins and proteoglycans. They are generally found on the exterior surface of cells. O- and N-linked glycans are very common in eukaryotes but may also be found, although less commonly, in prokaryotes. N-Linked glycans are found attached to the R-group nitrogen (N) of asparagine in the sequon. The sequon is a Asn-X-Ser or Asn-X-Thr sequence, where X is any amino acid except praline.

The term “exo-glycosylase” used herein refers to an enzyme capable of hydrolysis of glycan structure from the out most non-reducing end. Examples of suitable exo-glycosylase include, but are not limited to sialidase, galactosidase, alpha-fucosidase, alpha-mannosidase. The term “endo-glycosylase” used herein refers to an enzyme capable of hydrolysis of glycan structures randomly from the inner sits of whole glycan. Examples include, but are not limited to Endo-H, Endo-F3, Endo-F2, and Endo-F 1.

The term “antibody” (Ab) as used herein includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity. The term “immunoglobulin” (Ig) is used interchangeably with “antibody” herein.

The term “isolated antibody” used herein refers to an antibody substantially free from naturally associated molecules, i.e., the naturally associated molecules constituting at most 20% by dry weight of a preparation containing the antibody. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody is purified: (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie Blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, and HPLC.

A “human antibody” as used herein refers to an antibody naturally existing in humans, a functional fragment thereof, or a humanized antibody, i.e., a genetically engineered antibody a portion of which (e.g., a frame region or the Fc region) derives from a naturally-occurring human antibody.

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier “monoclonal” is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies useful in the present invention may be prepared by the hybridoma methodology first described by Kohler et al., Nature, 256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example.

The term “variable” refers to the fact that certain segments of the V domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and defines specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110-amino acid span of the variable domains. Instead, the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called “hypervariable regions” that are each 9-12 amino acids long. The variable domains of native heavy and light chains each comprise four FRs, largely adopting a β-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the β,β-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).

The term “hypervariable region” when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region generally comprises amino acid residues from a “complementarity determining region” or “CDR” (e.g., around about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the VL, and around about 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the VH when numbered in accordance with the Kabat numbering system; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)); and/or those residues from a “hypervariable loop” (e.g., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the VL, and 26-32 (H1), 52-56 (H2) and 95-101 (H3) in the VH when numbered in accordance with the Chothia numbering system; Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); and/or those residues from a “hypervariable loop”/CDR (e.g., residues 27-38 (L1), 56-65 (L2) and 105-120 (L3) in the VL, and 27-38 (H1), 56-65 (H2) and 105-120 (H3) in the VH when numbered in accordance with the IMGT numbering system; Lefranc, M. P. et al. Nucl. Acids Res. 27:209-212 (1999), Ruiz, M. e al. Nucl. Acids Res. 28:219-221 (2000)). Optionally the antibody has symmetrical insertions at one or more of the following points 28, 36 (L1), 63, 74-75 (L2) and 123 (L3) in the VL, and 28, 36 (H1), 63, 74-75 (H2) and 123 (H3) in the VH when numbered in accordance with Honneger, A. and Plukthun, A. J. Mol. Biol. 309:657-670 (2001)).

The monoclonal antibodies herein include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). The present invention provides variable domain antigen-binding sequences derived from human antibodies. Accordingly, chimeric antibodies of primary interest herein include antibodies having one or more human antigen binding sequences (e.g., CDRs) and containing one or more sequences derived from a non-human antibody, e.g., an FR or C region sequence. In addition, chimeric antibodies of primary interest herein include those comprising a human variable domain antigen binding sequence of one antibody class or subclass and another sequence, e.g., FR or C region sequence, derived from another antibody class or subclass. Chimeric antibodies of interest herein also include those containing variable domain antigen-binding sequences related to those described herein or derived from a different species, such as a non-human primate (e.g., Old World Monkey, Ape, etc). Chimeric antibodies also include primatized and humanized antibodies.

Furthermore, chimeric antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Opin. Struct. Biol. 2:593-596 (1992).

A “humanized antibody” is generally considered to be a human antibody that has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization is traditionally performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting import hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.

A “human antibody” is an antibody containing only sequences present in an antibody naturally produced by a human. However, as used herein, human antibodies may comprise residues or modifications not found in a naturally occurring human antibody, including those modifications and variant sequences described herein. These are typically made to further refine or enhance antibody performance.

An “intact” antibody is one that comprises an antigen-binding site as well as a CL and at least heavy chain constant domains, CH 1, CH 2 and CH 3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variant thereof. Preferably, the intact antibody has one or more effector functions.

An “antibody fragment” comprises a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain along with the variable region domain of the H chain (VH), and the first constant domain of one heavy chain (CH 1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab′)₂ fragment that roughly corresponds to two disulfide linked Fab fragments having divalent antigen-binding activity and is still capable of cross-linking antigen. Fab′ fragments differ from Fab fragments by having additional few residues at the carboxy terminus of the CH1 domain including one or more cysteines from the antibody hinge region. Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab′)₂ antibody fragments originally were produced as pairs of Fab′ fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

The “Fc” fragment comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, which region is also the part recognized by Fc receptors (FcR) found on certain types of cells.

“Fv” is the minimum antibody fragment that contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (three loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

An antibody having a “biological characteristic” of a designated antibody is one that possesses one or more of the biological characteristics of that antibody which distinguish it from other antibodies. For example, in certain embodiments, an antibody with a biological characteristic of a designated antibody will bind the same epitope as that bound by the designated antibody and/or have a common effector function as the designated antibody.

An “antibody that inhibits the growth of infected cells” or a “growth inhibitory” antibody is one that binds to and results in measurable growth inhibition of infected cells expressing or capable of expressing an HIV1 epitope bound by an antibody. Preferred growth inhibitory antibodies inhibit growth of infected cells by greater than 20%, preferably from about 20% to about 50%, and even more preferably, by greater than 50% (e.g., from about 50% to about 100%) as compared to the appropriate control, the control typically being infected cells not treated with the antibody being tested. Growth inhibition can be measured at an antibody concentration of about 0.1 to 30 μg/ml, or about 0.5 nM to 200 nM in cell culture, where the growth inhibition is determined 1-10 days after exposure of the infected cells to the antibody. Growth inhibition of infected cells in vivo can be determined in various ways known in the art. The antibody is growth inhibitory in vivo if administration of the antibody at about 1 μg/kg to about 100 mg/kg body weight results in reduction the percent of infected cells or total number of infected cells within about 5 days to 3 months from the first administration of the antibody, preferably within about 5 to 30 days.

An antibody that “induces apoptosis” is one which induces programmed cell death as determined by binding of annexin V, fragmentation of DNA, cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation, and/or formation of membrane vesicles (called apoptotic bodies). Preferably the cell is an infected cell. Various methods are available for evaluating the cellular events associated with apoptosis. For example, phosphatidyl serine (PS) translocation can be measured by annexin binding; DNA fragmentation can be evaluated through DNA laddering; and nuclear/chromatin condensation along with DNA fragmentation can be evaluated by any increase in hypodiploid cells. Preferably, the antibody that induces apoptosis is one that results in about 2 to 50 fold, preferably about 5 to 50 fold, and most preferably about 10 to 50 fold, induction of annexin binding relative to untreated cell in an annexin binding assay.

Antibody “effector functions” refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies “arm” the cytotoxic cells and are required for such killing. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA) 95:652-656 (1998).

“Fc receptor” or “FcR” describes a receptor that binds to the Fc region of an antibody. In certain embodiments, the FcR is a native sequence human FcR. Moreover, a preferred FcR is one that binds an IgG antibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA (an “activating receptor”) and FcγRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (reviewed by M. Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term “FcR” herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., Eur. J. Immunol. 24:2429-2434 (1994)).

“Human effector cells” are leukocytes that express one or more FcRs and perform effector functions. Preferably, the cells express at least FcγRIII and perform ADCC effector function. Examples of human leukocytes that mediate ADCC include PBMC, NK cells, monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK cells being preferred. The effector cells may be isolated from a native source, e.g., from blood.

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) that are bound to their cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1997), may be performed.

A “mammal” for purposes of treating an infection, refers to any mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is human.

“Treating” or “treatment” or “alleviation” refers to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. A subject or mammal is successfully “treated” for an infection if, after receiving a therapeutic amount of an antibody according to the methods of the present invention, the patient shows observable and/or measurable reduction in or absence of one or more of the following: reduction in the number of infected cells or absence of the infected cells; reduction in the percent of total cells that are infected; and/or relief to some extent, one or more of the symptoms associated with the specific infection; reduced morbidity and mortality, and improvement in quality of life issues. The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.

The term “therapeutically effective amount” refers to an amount of an antibody or a drug effective to “treat” a disease or disorder in a subject or mammal. See preceding definition of “treating.”

“Chronic” administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time. “Intermittent” administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.

Administration “in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

“Carriers” as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™ polyethylene glycol (PEG), and PLURONICS™.

The term “cytotoxic agent” as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactive isotopes of Lu), chemotherapeutic agents e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleolytic enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof, and the various antitumor or anticancer agents disclosed below. Other cytotoxic agents are described below.

A “growth inhibitory agent” when used herein refers to a compound or composition which inhibits growth of a cell, either in vitro or in vivo. Examples of growth inhibitory agents include agents that block cell cycle progression, such as agents that induce G1 arrest and M-phase arrest. Classic M-phase blockers include the vinca alkaloids (vincristine, vinorelbine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that arrest G1 also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation, oncogenes, and antineoplastic drugs” by Murakami et al. (W B Saunders: Philadelphia, 1995), especially p. 13. The taxanes (paclitaxel and docetaxel) are anticancer drugs both derived from the yew tree. Docetaxel (TAXOTERE™, Rhone-Poulenc Rorer), derived from the European yew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which results in the inhibition of mitosis in cells.

The term “polypeptide” is used in its conventional meaning, i.e., as a sequence of amino acids. The polypeptides are not limited to a specific length of the product. Peptides, oligopeptides, and proteins are included within the definition of polypeptide, and such terms may be used interchangeably herein unless specifically indicated otherwise. This term also does not refer to or exclude post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. A polypeptide may be an entire protein, or a subsequence thereof. Particular polypeptides of interest in the context of this invention are amino acid subsequences comprising CDRs and being capable of binding an antigen or HIV-infected cell.

An “isolated polypeptide” is one that has been identified and separated and/or recovered from a component of its natural environment. In preferred embodiments, the isolated polypeptide will be purified (1) to greater than 95% by weight of polypeptide as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. Isolated polypeptide includes the polypeptide in situ within recombinant cells since at least one component of the polypeptide's natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.

A polypeptide “variant,” as the term is used herein, is a polypeptide that typically differs from a polypeptide specifically disclosed herein in one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the above polypeptide sequences of the invention and evaluating one or more biological activities of the polypeptide as described herein and/or using any of a number of techniques well known in the art.

Antibodies of the Invention

The antibodies and antibody fragments of the invention are glycosylated in their Fc regions with oligosaccharides that are (a) free of core fucoses and/or (b) include terminal sialic acids linked to galactoses. Preferably, the oligosaccharides are homogeneous. In some aspects the antibodies are human or humanized. Where the Fc receptor binding protein is derived from the human IgG1 immunoglobulin or the human IgG3 immunoglobulin, the sequence of the Cγ2 domain present within the Fc receptor binding protein retains a highly-conserved N-linked glycosylation site at the asparagine (Asn, N) residue at position 297 (N297). The glycosylation of this residue has been identified as being important for mediating high affinity binding and activation of Fc receptors.

In one embodiment, the terminal sugar units of the invention are sialic acids linked to galactose. When the modified human or humanized antibodies are IgG molecules, the oligosaccharide moieties attached to the Fc regions can have the structures:

Disclosed herein is an antibody or antibody fragment in which both Ig domains comprise Fc regions that are attached to a monosaccharide moiety (e.g., N-Acetylglucosamine, GlcNAc) or a trisaccharide moiety (e.g., Mannose-N-Acetylglucosamine-N-Acetylglucosamine, Man-GlcNAc-GlcNAc).

Preferably, the trisaccharides are identical to the trisaccharide portions of the oligosaccharides attached to Fc regions of naturally-occurring human antibodies. When the antibodies are IgG molecules, the trisaccharides can have the structure of Man-GlcNAc-GlcNAc.

Any of the antibodies of this invention can be prepared from a commercially available therapeutic antibody (e.g., Reopro®, Rituxan®, Zenepax®, Simulect®, Synagis®, Remicade®, Herceptin®, Mylotarg®, Campath®, Zevalin®, Humira®, Xolair®, Bexxar®, Raptiva®, Erbitux®, Avastin®, Tysabri), human or humanized antibodies produced via a conventional method, preferably those undergoing clinical trials.

Other monoclonal antibodies suitable for this invention include, but are not limited to Cetuximab®, Rituximab®, Muromonab-CD3®, Abciximab®, Daclizumab®, Basiliximab®, Palivizumab®, Infliximab®, Trastuzumab®, Gemtuzumab Ozogamicin®, Alemtuzumab®, Ibritumomab Tiuxetan®, Adalimumab®, Omalizumab®, tositumomab®, 1-131 tositumomab®, Efalizumab®, Bevacizumab®, Panitumumab®, Pertuzumab®, Natalizumab®, Etanercept®, IGN101®, Volociximab®, Anti-CD80 Mab, Anti-CD23 Mab, CAT-3888®, CDP-791®, Eraptuzumab®, MDX-010®, MDX-060®, MDX-070®, Matuzumab®, CP-675®,206®, CAL®, SGN-30®, Zanolimumab®, Adecatumumab®, Oregovomab®, Nimotuzumab®, ABT-874®, Denosumab®, AM 108®, AMG 714®, Fontolizumab®, Daclizumab®, Golimumab®, CNTO 1275®, Ocrelizumab®, Humax-CD20®, Belimumab®, Epratuzumab®, MLN1202®, Visilizumab®, Tocilizumab®, Ocrerlizumab®, Certolizumab Pegol®, Eculizumab®, Pexelizumab®, Abciximab®, Ranibizimumab®, Mepolizumab®, TNX-355®, and MYO-029®.

The antibodies of the present invention may be polyclonal or monoclonal antibodies. However, in preferred embodiments, they are monoclonal. In particular embodiments, antibodies of the present invention are human antibodies. Methods of producing polyclonal and monoclonal antibodies are known in the art and described generally, e.g., in U.S. Pat. No. 6,824,780. Typically, the antibodies of the present invention are produced by recombinant techniques, using vectors and methods available in the art, as described further below. Human antibodies may also be generated by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).

Human antibodies may also be produced in transgenic animals (e.g., mice) that are capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (J_(H)) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array into such germ-line mutant mice results in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immunology, 7:33 (1993); U.S. Pat. Nos. 5,545,806, 5,569,825, 5,591,669 (all of GenPharm); 5,545,807; and WO 97/17852. Such animals may be genetically engineered to produce human antibodies comprising a polypeptide of the present invention.

In certain embodiments, antibodies of the present invention are chimeric antibodies that comprise sequences derived from both human and non-human sources. In particular embodiments, these chimeric antibodies are humanized or primatized™. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

The choice of non-human sequences, both light and heavy, to be used in making the chimeric antibodies is important to reduce antigenicity and human anti-non-human antibody responses when the antibody is intended for human therapeutic use. It is further important that chimeric antibodies retain high binding affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, chimeric antibodies are prepared by a process of analysis of the parental sequences and various conceptual chimeric products using three-dimensional models of the parental human and non-human sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

As noted above, antibodies (or immunoglobulins) can be divided into five different classes, based on differences in the amino acid sequences in the constant region of the heavy chains. All immunoglobulins within a given class have very similar heavy chain constant regions. These differences can be detected by sequence studies or more commonly by serological means (i.e. by the use of antibodies directed to these differences). Antibodies, or fragments thereof, of the present invention may be any class, and may, therefore, have a gamma, mu, alpha, delta, or epsilon heavy chain. A gamma chain may be gamma 1, gamma 2, gamma 3, or gamma 4; and an alpha chain may be alpha 1 or alpha 2.

In a preferred embodiment, an antibody of the present invention, or fragment thereof, is an IgG. IgG is considered the most versatile immunoglobulin, because it is capable of carrying out all of the functions of immunoglobulin molecules. IgG is the major Ig in serum, and the only class of Ig that crosses the placenta. IgG also fixes complement, although the IgG4 subclass does not. Macrophages, monocytes, PMN's and some lymphocytes have Fc receptors for the Fc region of IgG. Not all subclasses bind equally well; for example, IgG2 and IgG4 do not bind to Fc receptors. A consequence of binding to the Fc receptors on PMN's, monocytes and macrophages is that the cell can now internalize the antigen better. IgG is an opsonin that enhances phagocytosis. Binding of IgG to Fc receptors on other types of cells results in the activation of other functions. Antibodies of the present invention may be of any IgG subclass.

In another preferred embodiment, an antibody, or fragment thereof, of the present invention is an IgE. IgE is the least common serum Ig since it binds very tightly to Fc receptors on basophils and mast cells even before interacting with antigen. As a consequence of its binding to basophils and mast cells, IgE is involved in allergic reactions. Binding of the allergen to the IgE on the cells results in the release of various pharmacological mediators that result in allergic symptoms. IgE also plays a role in parasitic helminth diseases. Eosinophils have Fc receptors for IgE and binding of eosinophils to IgE-coated helminths results in killing of the parasite. IgE does not fix complement.

As noted above, the present invention further provides antibody fragments comprising a polypeptide of the present invention. In certain circumstances there are advantages of using antibody fragments, rather than whole antibodies. For example, the smaller size of the fragments allows for rapid clearance, and may lead to improved access to certain tissues, such as solid tumors. Examples of antibody fragments include: Fab, Fab′, F(ab′)₂ and Fv fragments; diabodies; linear antibodies; single-chain antibodies; and multispecific antibodies formed from antibody fragments.

Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells.

In certain embodiments, antibodies of the present invention are bispecific or multi-specific. Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of a single antigen. Other such antibodies may combine a first antigen binding site with a binding site for a second antigen. Alternatively, an anti-HIV1 arm may be combined with an arm that binds to a triggering molecule on a leukocyte, such as a T-cell receptor molecule (e.g., CD3), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16), so as to focus and localize cellular defense mechanisms to the infected cell. Bispecific antibodies may also be used to localize cytotoxic agents to infected cells. These antibodies possess an HIV1-binding arm and an arm that binds the cytotoxic agent (e.g., saporin, anti-interferon -α, vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten). Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F(ab′)₂ bispecific antibodies). WO 96/16673 describes a bispecific anti-ErbB²/anti-FcγRIII antibody and U.S. Pat. No. 5,837,234 discloses a bispecific anti-ErbB2/anti-FcγRI antibody. A bispecific anti-ErbB2/Fcα antibody is shown in WO98/02463. U.S. Pat. No. 5,821,337 teaches a bispecific anti-ErbB2/anti-CD3 antibody.

According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. Preferably, the fusion is with an Ig heavy chain constant domain, comprising at least part of the hinge, C_(H)2, and C_(H)3 regions. It is preferred to have the first heavy-chain constant region (C_(H)1) containing the site necessary for light chain bonding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host cell. This provides for greater flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yield of the desired bispecific antibody. It is, however, possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios have no significant affect on the yield of the desired chain combination.

Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tuft et al., J. Immunol. 147: 60 (1991). A multivalent antibody may be internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind. The antibodies of the present invention can be multivalent antibodies with three or more antigen binding sites (e.g., tetravalent antibodies), which can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody. The multivalent antibody can comprise a dimerization domain and three or more antigen binding sites. The preferred dimerization domain comprises (or consists of) an Fc region or a hinge region. In this scenario, the antibody will comprise an Fc region and three or more antigen binding sites amino-terminal to the Fc region. The preferred multivalent antibody herein comprises (or consists of) three to about eight, but preferably four, antigen binding sites. The multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain(s) comprise two or more variable domains. For instance, the polypeptide chain(s) may comprise VD1-(X1)_(n)-VD2-(X2)_(n)-Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, X1 and X2 represent an amino acid or polypeptide, and n is 0 or 1. For instance, the polypeptide chain(s) may comprise: V_(H)-C_(H)1-flexible linker-V_(H)-C_(H)1-Fc region chain; or V_(H)-C_(H)1-V_(H)-C_(H)1-Fc region chain. The multivalent antibody herein preferably further comprises at least two (and preferably four) light chain variable domain polypeptides. The multivalent antibody herein may, for instance, comprise from about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptides contemplated here comprise a light chain variable domain and, optionally, further comprise a C_(L) domain. Antibodies of the present invention further include single chain antibodies.

Methods for preparing any of the human antibodies described above are disclosed.

In one example, this method includes (i) providing a human antibody, which is glycosylated in the Fc region; and (ii) contacting the antibody with alpha-fucosidase, alpha-2,6-sialyltransferase, and optionally β-1,4-galactosyltransferase to modify the oligosaccharides contained in the antibody, thereby producing a human antibody of this invention. When both β-1,4-galactosyltransferase and alpha-2,6-sialyltransferase are used, the antibody to be modified must first contact with the β-1,4-galactosyltransferase and then the alpha-2,6-sialyltransferase. Alpha-2,6-sialytransferase and β-galactosyltransferase transfer a sialic acid and a galactose, respectively, to an oligosaccharide via a glycosidic bond. In one example, the alpha-fucosidase and one of the β-1,4-galactosyltransferase and alpha-2,6-sialyltransferase can be immobilized on a support member (e.g., a bead). FIG. 1 shows a schematic illustration depicting the pathway (Path A)

FIG. 2 shows a schematic illustration depicting a chemoenzymatic pathway (Path B) for preparing human antibodies with a single glycoform by glycan engineering, using various exo-glycosidases, endo-glycosidases, and glycosyltransferases. The method the method comprises: (i) providing a Fc region of an antibody or antibody fragment, wherein the Fc region is glycosylated with an oligosaccharide; (ii) contacting the Fc region with an endo-glycosylase (such as endo-H, endo-F3, etc.) and an exo-glycosylase (such as sialidase, galactosidase, alpha-fucosidase, or a mixture thereof) under conditions wherein the oligosaccharide is digested to a single sugar unit (such as GlcNAc); (iii) elongating the single sugar unit to an oligosaccharide by glycosylation mediated by one or more glycosyltransferases such as endo-N-acetylglucosaminidase, endo-M or endo-A; and (iv) contacting the oligosaccharide with alpha-2,6-sialytransferase to add a terminal sialic acid, thereby yielding the antibody having homogeneous oligosaccharides.

The oligosaccharide obtained from the elongating step can have a sugar sequence identical to at least a portion of an oligosaccharide found in a naturally-occurring antibody, i.e., a whole Ig molecule produced in a cell (either in cell culture or in a host animal) that glycosylates the Ig molecule in its Fc region. When the antibody is an IgG, the oligosaccharide can have the structure of:

In yet another example, the method of this invention comprises five steps: (i) providing a Fc region of an antibody or antibody fragment, wherein the Fc region is glycosylated with an oligosaccharide; (ii) contacting the Fc region with an endo-glycosylase (e.g., endo-H, endo-F3, etc.) and an exo-glycosylase (e.g., sialidase, galactosidase, alpha-fucosidase, or a mixture thereof) under conditions wherein the oligosaccharide is digested to a single sugar unit (e.g., GlcNAc); (iii) elongating the single sugar unit to a first oligosaccharide via glycosylation mediated by glycosyltransferases such as endo-N-acetylglucosaminidase, endo-M or endo-A; (iv) contacting the first oligosaccharide with a β-1,4-galactosyltransferase to produce a second oligosaccharide having a terminal galactose, and (v) contacting the second oligosaccharide with alpha-2,6-sialytransferase to add a terminal sialic acid, thereby yielding antibody populations having Fc regions glycosylated with homogeneous oligosaccharides.

In still another example, the method of this invention is performed as follows: (i) providing a human antibody, the Fc region of which is attached to oligosaccharides; (ii) treating the antibody with an exo-glycosylase (e.g., alpha-mannosidase, alpha-fucosidase, sialidase, galactosidase, or a mixture thereof) to trim each of the oligosaccharides to a trisaccharide (e.g., ManGlcNAcGlcNAc); (iii) elongating the trisaccharide to an oligosaccharide via glycosylation; and (iv) treating the oligosaccharide with alpha-2,6-sialytransferase to add terminal sialic acids, thereby yielding the antibody in a single glycoform. FIG. 3 shows a schematic illustration depicting such an enzymatic pathway (Path C).

Preferably, the oligosaccharide obtained from the elongating step has a sugar sequence identical to a portion of an oligosaccharide found in the Fc region of a naturally occurring human antibody. When the antibody is an IgG, the oligosaccharide can have the structure of:

The oligosaccharide is then subjected to alpha-2,6-sialytransferase treatment to add terminal sialic acid residues.

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLES Example 1 Method A for Making Antibodies of the Invention

Following Path A depicted in FIG. 1, a human antibody can be treated with α-fucosidase to remove core fucoses and with α-2,6-siayltransferase to add terminal α-2,6-linked sialic acids. When necessary, it can be treated with β-1,4-galactosyltransferase to add a galactose and then with α-2,6-siayltransferase to add the terminal α-2,6-linked sialic acids. Preferably, the enzymes used in this method can be immobilized on a support member. In one example, α-fucosidase and α-2,6-siayltransferase are coated onto a plurality of beads and the resultant coated beads are packed in a column. A sample containing an antibody, either obtained from a commercial vendor or produced by a conventional method, is then loaded onto the column under conditions suitable for the enzymatic reactions catalyzed by the α-fucosidase and α-2,6-siayltransferase. The antibody, with core fucose removed and terminal sialic acid added, is then eluted by a suitable solution.

Example 2 Method B for Making Antibodies of the Invention

Alternatively, an antibody of this invention can be prepared following Path B described in FIG. 2. FIG. 2 shows an example for preparing an IgG antibody with its Fc region glycosylated with biantennary N-linked oligosaccharides. A human IgG antibody, having a glycosylated Fc region, was treated with sialidase and galactosidase, followed by Endo-H and alpha-fucosidase to produce an antibody having both Ig domains in the Fc region attached to a single GlcNAc residue. The antibody having two single GlcNAc residues attached (one in each of the Ig domains) is then subjected to endoglycosidase-catalyzed transglycosylation to elongate each of the GlcNAc residues to an oligosaccharide. In one example, a synthetic oligosaccharide with suitable leaving group like —OH, F or others is added to the GlcNAc mono-saccharide on the modified antibody via a glycosidase enzyme. Another example is a synthetic oligosaccharide with oxazoline group is added to the GlcNAc residue via an endo-beta-N-acetylglucosaminidase (NAG), endo-A or endo-M-mediated enzymatic reaction. The oligosaccharide is then treated with α-2,6-siayltransferase in the presence of CMP-Neu5Ac to add a terminal sialic acid residues. When necessary, a galactose residue is added to the oligosaccharide via β-1,4-galactosyltransferase, using UDP-Gal as a substrate, before addition of the sialic acids.

Example 3 Method C for Making Antibodies of the Invention

An antibody of this invention can also be prepared following the process (i.e., Path C) depicted in FIG. 3. In this process, a human antibody with glycosylated Fc region can be first treated with one or more exo-glycosylases to remove core fucoses and to trim the original oligosaccharides attached to the Fc region to trisaccharides. The core fucose-free trisaccharides are then elongated by glysocyltransferases to desired oligosaccharides, e.g., having sugar sequences identical to those found in naturally occurring human antibodies. Finally, the desired oligosaccharides are treated with β-1,4-galactosyltrasferase and α-2,6-siayltransferase sequentially to add galactose residues and then terminal sialic acid residues.

Example 4 Methods for Cleaving the Chitobiose Linkage of Fc N-Linked Glycans

Endo F2 and Endo F3 are unique in their ability to cleave complex structures. Endo F2 cleaves asparagine-linked or free oligomannose, and biantennary complex oligosaccharides. Oligomannose structures are cleaved at a 20-fold reduced rate. Fucosylation has little effect on Endo F2 cleavage of biantennary structures. It will not cleave hybrid structures. It cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine.

Endo F3 is unique in that its cleavage is sensitive to the state of peptide linkage of the oligosaccharide, as well as the state of core fucosylation. Endoglycosidase F3 cleaves asparagine-linked biantennary and triantennary complex oligosaccharides. It will cleave non-fucosylated biantennary and triantennary structures at a slow rate, but only if peptide-linked. Core fucosylated biantennary structures are efficient substrates for Endo F3, even as free oligosaccharides. Endo F3 will also cleave fucosylated trimannosyl core structures on free and protein-linked oligosaccharides.

As shown in FIG. 5A, Humira® obtained from CHO cells, comprises a heterogeneous population of glycoforms with a predominance of the fucosylated glycoform (GlcNAc-Man)2-Man-GlcNAc-GlcNAc(Fucose)-[Fc].

Digestion with Endo F2 (in 0.05M NaH₂PO₄, pH 4.54 buffer) or Endo F3 (in 0.05M CH₃COONa, pH 4.54 buffer) yielded a glycoform with a single fucosylated GlcNAc as visualized by 8-16% polyacrylamide gel electrophoresis in Tris-glycine buffer with Coomassie Blue stain (FIG. 5B)

Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are encompassed within the scope of the claimed invention.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. 

What is claimed is:
 1. A method for glycan engineering the Fc region of a human, chimeric or humanized IgG1 or IgG3 glycosylated antibody, the method comprising the steps of: (a) providing a human, chimeric or humanized IgG1 or IgG3 glycosylated antibody comprising an oligosaccharide attached at each Asn 297 in the Fc region; (b) contacting the human, chimeric or humanized IgG1 or IgG3 glycosylated antibody with an endo-β-N-acetylglucosaminidase (endo-NAG) and alpha-fucosidase thereby digesting the oligosaccharide to a defucosylated N-Acetylglucosamine (GlcNAc) unit attached at each Asn 297 in the Fc region; (c) contacting the defucosylated N-Acetylglucosamine (GlcNAc) unit with an endo-β-N-acetylglucosaminidase (endo-NAG) and a synthetic Gal₂GlcNAc₂Man₃GlcNAc moiety joined to an oxazoline group, thereby coupling the terminal GlcNac of the synthetic Gal₂GlcNAc₂Man₃GlcNAc moiety to the defucosylated N-Acetylglucosamine (GlcNAc) unit to form the structure Gal₂GlcNAc₂Man₃GlcNAc₂ attached at each Asn 297 of the Fc region of the human, chimeric or humanized IgG1 or IgG3 glycosylated antibody; and (d) contacting the attached Gal₂GlcNAc₂Man₃GlcNAc₂ of step (c) with an alpha-2,6-sialyltransferase, CMP-Neu5Ac and a set of CMP-Neu5Ac regeneration enzymes comprising a pyrophosphatase and a cytidine monophosphate kinase, thereby adding a terminal Neu5Ac to each terminal galactose of the attached Gal₂GlcNAc₂Man₃GlcNAc₂, wherein the glycan engineered human, chimeric or humanized IgG1 or IgG3 antibody has a Neu5Ac₂Gal₂GlcNAc₂Man₃GlcNAc₂ attached at each Asn 297 in the Fc region, and wherein the CMP-Neu5Ac is regenerated following the addition of the terminal Neu5Ac.
 2. The method of claim 1, wherein the human, chimeric or humanized IgG1 or IgG3 glycosylated antibody is selected from the group consisting of Abciximab, Rituximab, Daclizumab, Basiliximab, Palivizumab, Infliximab, Trastuzumab, Alemtuzumab, Ibritumomab Tiuxetan, Ibritumomab Tiuxetan, Omalizumab, Tositumomab, Efalizumab, Cetuximab, and Bevacizumab.
 3. The method of claim 1, wherein the endo-β-N-acetylglucosaminidase (endo-NAG) and alpha-fucosidase are immobilized on a solid support.
 4. The method of claim 3, wherein the solid support is a bead.
 5. The method of claim 1, wherein the endo-β-N-acetylglucosaminidase (endo-NAG) in step (b) is selected from the group consisting of: Endo H, Endo A, Endo M, Endo F2, and Endo F3.
 6. The method of claim 1, wherein the endo-β-N-acetylglucosaminidase (endo-NAG) is Endo H.
 7. The method of claim 1, wherein the synthetic Gal₂GlcNAc₂Man₃GlcNAc moiety comprising terminal galactose units has the following structure:

wherein X is an oxazoline group.
 8. The method of claim 2, wherein the human, chimeric or humanized IgG1 or IgG3 glycosylated antibody is Rituximab.
 9. A method for glycan engineering the Fc region of a human, chimeric or humanized glycosylated IgG1 or IgG3 antibody, the method comprising the steps of: (a) providing a human, chimeric or humanized IgG1 or IgG3 glycosylated antibody comprising an oligosaccharide attached at each Asn 297 in the Fc region; (b) contacting the human, chimeric or humanized IgG1 or IgG3 glycosylated antibody with an endo-β-N-acetylglucosaminidase (endo-NAG) and alpha-fucosidase thereby digesting the oligosaccharide to a defucosylated N-Acetylglucosamine (GlcNAc) unit attached at each Asn 297 in the Fc region; (c) contacting the defucosylated N-Acetylglucosamine (GlcNAc) unit with an endo-β-N-acetylglucosaminidase (endo-NAG) and a synthetic GlcNAc₂Man₃GlcNAc moiety joined to a leaving group, thereby coupling the terminal GlcNac unit of the synthetic oligosaccharide moiety to the single GlcNAc unit of the synthetic GlcNAc₂Man₃GlcNAc moiety to the defucosylated N-Acetylglucosamine (GlcNAc) unit to form the structure GlcNAc₂Man₃GlcNAc₂ attached at each Asn 297 of the Fc region of the human, chimeric or humanized IgG1 or IgG3 glycosylated antibody; and (d) contacting the attached GlcNAc₂Man₃GlcNAc₂ of step (c) with a β-1,4-galactosyltransferase, UDP galactose, and a set of UDP-galactose regeneration enzymes selected from the group consisting of a glucose kinase, a phosphoglucomutase, an N-acetyl glucosamine-1 phosphate uridyltransferase, a polyphosphate kinase, a pyrophosphatase, a nucleoside diphosphate kinase, a UDP-Glc/GlcNAc₄ epimerase, a pyruvate kinase, a UDP kinase, a galactokinase, an ADP kinase, and a UDP-galactose pyrophosphorylase. thereby adding a terminal galactose to each terminal GlcNAc of the attached GlcNAc₂Man₃GlcNAc₂ and wherein the UDP-galactose is regenerated following the addition of the terminal galactose; and (e) contacting the attached Gal₂GlcNAc₂Man₃GlcNAc₂ of step (d) with an alpha-2,6-sialyltransferase, CMP-Neu5Ac, and a set of CMP-Neu5Ac regeneration enzymes comprising a pyrophosphatase and a cytidine monophosphate kinase, thereby adding a terminal Neu5Ac to each terminal galactose of the attached Gal₂GlcNAc₂Man₃GlcNAc₂, wherein the glycan engineered human, chimeric or humanized IgG1 or IgG3 antibody has a Neu5Ac₂Gal₂GlcNAc₂Man₃GlcNAc₂ attached at each Asn 297 of the Fc region and wherein the CMP-Neu5Ac is regenerated following the addition of the terminal Neu5Ac.
 10. The method of claim 9, wherein the endo-β-N-acetylglucosaminidase (endo-NAG) and alpha-fucosidase are immobilized on a solid support.
 11. The method of claim 10, wherein the solid support is a bead.
 12. The method of claim 9, wherein the endo-β-N-acetylglucosaminidase (endo-NAG) in step (b) is selected from the group consisting of: Endo H, Endo A, Endo M, Endo F2, and Endo F3.
 13. The method of claim 9 wherein the endo-β-N-acetylglucosaminidase (endo-NAG) is Endo H.
 14. The method of claim 9, wherein the leaving group is selected from the group consisting of: fluoride (F), OH (hydroxy), and oxazoline.
 15. The method of claim 9, wherein the synthetic GlcNAc₂Man₃GlcNAc moiety has the following structure:

wherein X is a leaving group.
 16. The method of claim 9, wherein the human, chimeric or humanized IgG1 or IgG3 glycosylated antibody is selected from the group consisting of Abciximab, Rituximab, Daclizumab, Basiliximab, Palivizumab, Infliximab, Trastuzumab, Alemtuzumab, Ibritumomab Tiuxetan, Ibritumomab Tiuxetan, Omalizumab, Tositumomab, Efalizumab, Cetuximab, and Bevacizumab.
 17. A method for glycan engineering the Fc region of a human, chimeric, or humanized glycosylated antibody, wherein the glycosylated antibody comprises an oligosaccharide attached at Asn-297, wherein the oligosaccharide is selected from the group consisting of Gal₂GlcNAc₂Man₃GlcNAc₂, GalGlcNAc₂Man₃GlcNAc₂, and GlcNAc₂Man₃GlcNAc₂, the method comprising: (a) contacting the antibody with an alpha fucosidase and β-1,4-galactosyltransferase and UDP galactose thereby defucosylating the oligosaccharide and coupling the terminal GlcNac of the GalGlcNAc₂Man₃GlcNAc₂, and the GlcNAc₂Man₃GlcNAc₂ to the galactose to form the structure Gal₂GlcNAc₂Man₃GlcNAc₂ attached at each Asn 297 of the Fc region of the human, chimeric or humanized glycosylated antibody; and (b) contacting the antibody comprising the structure Gal₂GlcNAc₂Man₃GlcNAc₂ with an alpha-2,6-sialyltransferase and CMP-Neu5Ac, thereby adding a terminal Neu5Ac to each terminal galactose of the Gal₂GlcNAc₂Man₃GlcNAc₂, wherein the glycan engineered human, chimeric or humanized antibody has a Neu5Ac₂Gal₂GlcNAc₂Man₃GlcNAc₂ attached at each Asn 297 in the Fc region.
 18. The method of claim 17, wherein the alpha fucosidase is immobilized on a bead
 19. The method of claim 17, wherein the β-1,4-galactosyltransferase is immobilized on a bead
 20. The method of claim 17, wherein the alpha-2,6-sialyltransferase is immobilized on a bead.
 21. The method of claim 17, further comprising a set of CMP-Neu5Ac regeneration enzymes comprising a pyrophosphatase and a cytidine monophosphate kinase, and wherein the CMP-Neu5Ac is regenerated following the addition of the terminal Neu5Ac.
 22. The method of claim 17, wherein the human, chimeric or humanized glycosylated antibody is selected from the group consisting of Abciximab, Rituximab, Daclizumab, Basiliximab, Palivizumab, Infliximab, Trastuzumab, Alemtuzumab, Ibritumomab Tiuxetan, Ibritumomab Tiuxetan, Omalizumab, Tositumomab, Efalizumab, Cetuximab, and Bevacizumab.
 23. A method for glycan engineering the Fc region of a human, chimeric, or humanized glycosylated antibody, wherein the glycosylated antibody comprises an oligosaccharide attached at Asn-297, wherein the oligosaccharide is selected from the group consisting of Gal₂GlcNAc₂Man₃GlcNAc₂, GalGlcNAc₂Man₃GlcNAc₂, and GlcNAc₂Man₃GlcNAc₂, the method comprising: (a) contacting the antibody with a β-1,4-galactosyltransferase and UDP galactose thereby coupling the terminal GlcNac of the GalGlcNAc₂Man₃GlcNAc₂, and the GlcNAc₂Man₃GlcNAc₂ to the galactose to form the structure Gal₂GlcNAc₂Man₃GlcNAc₂ attached at each Asn 297 of the Fc region of the human, chimeric or humanized glycosylated antibody; and (b) contacting the antibody comprising the structure Gal₂GlcNAc₂Man₃GlcNAc₂ with an alpha-2,6-sialyltransferase and CMP-Neu5Ac, thereby adding a terminal Neu5Ac to each terminal galactose of the Gal₂GlcNAc₂Man₃GlcNAc₂, wherein the glycan engineered human, chimeric or humanized antibody has a Neu5Ac₂Gal₂GlcNAc₂Man₃GlcNAc₂ attached at each Asn 297 in the Fc region.
 24. The method of claim 23, wherein the β-1,4-galactosyltransferase and the alpha-2,6-sialyltransferase are immobilized on a solid support.
 25. The method of claim 24, wherein the solid support is a bead.
 26. The method of claim 23, further comprising contacting the antibody of step (a) having the structure structure Gal₂GlcNAc₂Man₃GlcNAc₂ attached at each Asn 297 of the Fc region with an alpha-fucosidase.
 27. The method of claim 26 wherein the alpha-fucosidase is immobilized on a solid support.
 28. The method of claim 27, wherein the solid support is a bead.
 29. The method of claim 23, further comprising a set of CMP-Neu5Ac regeneration enzymes comprising a pyrophosphatase and a cytidine monophosphate kinase, and wherein the CMP-Neu5Ac is regenerated following the addition of the terminal Neu5Ac.
 30. The method of claim 23, wherein the human, chimeric, or humanized glycosylated antibody comprises an IgG or an IgE.
 31. The method of claim 30, wherein the IgG is an IgG1, an IgG2, and IgG3, or an IgG4.
 32. The method of claim 23, wherein the human, chimeric or humanized glycosylated antibody is selected from the group consisting of Abciximab, Rituximab, Daclizumab, Basiliximab, Palivizumab, Infliximab, Trastuzumab, Alemtuzumab, Ibritumomab Tiuxetan, Ibritumomab Tiuxetan, Omalizumab, Tositumomab, Efalizumab, Cetuximab, and Bevacizumab.
 33. A method for glycan engineering the Fc region of a human, chimeric, or humanized glycosylated antibody, the method comprising: (a) providing an antibody comprising an N-glycan selected from the group consisting of Gal₂GlcNAc₂Man₃GlcNAc, GalGlcNAc₂Man₃GlcNAc, and GlcNAc₂Man₃GlcNAc, attached at Asn-297; (b) contacting the antibody of step (a) with β-1,4-galactosyltransferase and UDP galactose (uridine diphosphate galactose) thereby coupling the terminal GlcNac of the GalGlcNAc₂Man₃GlcNAc, and the GlcNAc₂Man₃GlcNAc to the galactose to form the structure Gal₂GlcNAc₂Man₃GlcNAc attached at each Asn 297 of the Fc region of the human, chimeric or humanized glycosylated antibody; and (c) contacting the antibody comprising the structure Gal₂GlcNAc₂Man₃GlcNAc₂ with an alpha-2,6-sialyltransferase and CMP-Neu5Ac, thereby adding a terminal Neu5Ac to each terminal galactose of the Gal₂GlcNAc₂Man₃GlcNAc₂, wherein the glycan engineered human, chimeric or humanized antibody has a Neu5Ac₂Gal₂GlcNAc₂Man₃GlcNAc₂ attached at each Asn 297 in the Fc region.
 34. The method of claim 33, wherein the β-1,4-galactosyltransferase and the alpha-2,6-sialyltransferase are immobilized on a solid support.
 35. The method of claim 34, wherein the solid support is a bead.
 36. The method of claim 33, further comprising contacting the antibody of step (a) having the structure structure Gal₂GlcNAc₂Man₃GlcNAc₂ attached at each Asn 297 of the Fc region with an alpha-fucosidase.
 37. The method of claim 36 wherein the alpha-fucosidase is immobilized on a solid support.
 38. The method of claim 37, wherein the solid support is a bead.
 39. The method of claim 33, further comprising a set of CMP-Neu5Ac regeneration enzymes comprising a pyrophosphatase and a cytidine monophosphate kinase, and wherein the CMP-Neu5Ac is regenerated following the addition of the terminal Neu5Ac.
 40. The method of claim 33, wherein the human, chimeric, or humanized glycosylated antibody comprises an IgG or an IgE.
 41. The method of claim 40, wherein the IgG is an IgG1, an IgG2, and IgG3, or an IgG4.
 42. The method of claim 33, wherein the human, chimeric or humanized glycosylated antibody is selected from the group consisting of Abciximab, Rituximab, Daclizumab, Basiliximab, Palivizumab, Infliximab, Trastuzumab, Alemtuzumab, Ibritumomab Tiuxetan, Ibritumomab Tiuxetan, Omalizumab, Tositumomab, Efalizumab, Cetuximab, and Bevacizumab. 